Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and recent studies have established a strong relationship between intramyocellular triglyceride accumulation and insulin resistance in skeletal muscle. In this proposal the mechanism of fatty acid induced insulin resistance in skeletal muscle will be examined in awake rats and unique transgenic and knockout mouse models using state-of-the-art methodology including nuclear magnetic resonance spectroscopy (NMR), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) in combination with radioactive isotope techniques. Recent studies by our group have demonstrated that fatty acids induce insulin resistance in skeletal muscle by inhibiting insulin activation of IRS-1 associated phosphatidylinositol 3-kinase (PI 3-kinase) which we hypothesized was mediated by activation of a serine kinase cascade involving protein kinase C? (PKC?). This grant will further examine this hypothesis by examining the sequence of events leading to activation of PKC? in skeletal muscle. Specifically, increases in intramuscular content of fatty acyl CoA, ceramides, diacylglycerol, and triglyceride in relation to activation of PKC?, IRS-1 Ser307 phosphorylation and insulin simulated: 1) insulin receptor tyrosine phosphorylation, 2) IRS-1 tyrosine phosphorylation and 3) IRS-1 associated PI 3-kinase activity will be examined in awake rats during a lipid infusion designed to raise plasma fatty acid concentrations. In order to examine the putative roles of PKC? and Jun kinase 1 in mediating fatty acid induced insulin resistance, the effect of lipid infusion and high-fat feeding on insulin action and signaling in skeletal muscle will be examined in PKC? and Jun kinase 1 knockout mice. To test the hypothesis that accumulation of intramyocellular fatty acid metabolites are responsible for mediating insulin resistance in skeletal muscle, insulin signaling and action will be examined in UCP3 overexpressing mice (to promote increased muscle fatty acid oxidation) and fatty acid transport 1 (FATP1) gene knockout mice (to block fatty acid entry into skeletal muscle) following lipid infusion and high-fat feeding. Finally, in order to test the hypothesis that n-3 fatty acids protect against fat induced insulin resistance by serving as natural ligands for PPAR[unreadable] resulting in peroxisome proliferation and increased hepatic fatty acid oxidation, the effect of feeding a diet enriched with n-3 fatty acids versus isocaloric control and safflower (% fat matched) diets on insulin signaling and action in PPAR[unreadable] knockout and wild type littermates will be examined. The results from these studies should yield important new insights into the mechanism of fatty acid induced insulin resistance in skeletal muscle leading to the identification of potential novel targets for treatment of type 2 diabetes.